OVERALL ? APOBEC MUTAGENESIS IN BREAST CANCER ABSTRACT APOBEC signature mutations make up 20% of base-substitution mutations in primary tumors, which increases to over 50% in metastases. Additional enrichment is often observed in estrogen receptor (ER)- positive disease. APOBEC-catalyzed C-to-U lesions in single-stranded (ss)DNA lead to signature C-to-T and C-to-G mutations within 5?-TCA and 5?-TCT trinucleotide motifs. In addition, APOBEC-derived C-to-U lesions can be (mis)processed by cellular DNA repair enzymes, resulting in single- and double-stranded DNA breaks and more complex chromosomal rearrangements. APOBEC expression levels and mutagenesis correspond with poor clinical outcomes, such as shorter disease-free and overall survival in women with operable ER- positive breast cancer. Elevated APOBEC levels also predict poor overall survival for patients diagnosed with recurrent ER-positive metastases. These and other published data demonstrate that APOBEC mutagenesis is ongoing in breast tumor cells and underpin our overarching Program hypothesis that inhibiting APOBEC will prevent a large proportion of additional mutations from happening in residual ER-positive disease and will thereby improve the durability of current treatments and result in better overall therapeutic outcomes. Three multidisciplinary Projects will work together in an integrated and comprehensive manner to test this idea. Project 1 will develop reporter systems for quantifying APOBEC activity in living cells and determine the molecular mechanisms responsible for APOBEC regulation and for genomic uracil processing in breast cancer cells. Project 2 will use chemical biology approaches to investigate the mechanism of APOBEC-catalyzed ssDNA deamination and will develop nucleic acid and small molecule probes to inhibit APOBEC activity. Project 3 will leverage structural and biophysical approaches to investigate global mechanisms for APOBEC binding to ssDNA as well as the local structural features important for target sequence preferences and inhibition of APOBEC enzymes in breast cancer. These Projects will be supported by service Cores for administration, murine models, computational chemistry and biophysics, and enzymes and antibodies. Our Program is poised to have both immediate and long-term impact for ER-positive breast cancer: immediate impact by producing novel technologies and a comprehensive understanding of the mechanism of APOBEC mutagenesis, and long-term impact on clinical translation through the development of technologies for diagnosing APOBEC-positive disease and the creation of novel chemical matter to inhibit this mutational process for therapeutic benefit.